vWF is a blood coagulation factor that is produced in a vascular endothelial cell and a megakaryocyte and is present as a multimer structure (with a molecular weight of 500 to 20,000 kDa) where single subunits each composed of 2050 amino acid residues (approximately 250-kDa monomers) are bound through a S—S bond. The concentration of vWF in blood is approximately 10 μg/ml, and in general, vWF with a higher molecular weight has higher specific activity.
The vWF has two major functions as a blood coagulation factor, one of which is a function as a carrier protein that binds to and thereby stabilizes blood coagulation factor VIII and another of which is a function of helping platelets adhere and aggregate to the tissue beneath vascular endothelial cells of injured vascular walls to form platelet thrombi.
Thrombotic thrombocytopenic purpura (hereinafter, also referred to as TTP) is a disease that causes platelet thrombi in body tissue arterioles and capillary vessels in the whole body. In spite of the current progression of medical technology, the mortality associated with the disease has increased approximately threefold from 1971 to 1991. Pathologically, the TTP is considered to be caused by vascular endothelia cell injury and platelet aggregation in blood vessels. Immunohistologically, the presence of vWF in large amounts is observed in generated platelet thrombi, and the vWF is considered to play a crucial role in the pathogenesis of the disease. TTP is broadly divided into familial (congenital) TTP likely to have an inheritance factor and acquired (idiopathic) TTP developed especially in adults. Normal or high-molecular-weight vWF multimer structures are dominant in TTP patients. Especially, unusually large vWF multimer (ULvWFM) and large vWF multimer (LvWFM) are presumed to play a crucial role in the promotion of platelet aggregation and microthrombus formation under high shearing stress. On the other hand, vWF has been known to undergo digestion at the location between 842Tyr and 843Met by the action of vWF-cleaving protease under high shearing stress in circulating blood of healthy individuals. Thus, a probable scenario of how TTP is caused is as follows: the activity of the protease in plasma is reduced for some reason and ULvWFM or LvWFM is increased to accelerate platelet aggregation, followed by platelet thrombus formation in the blood vessel.
In 2001, a gene encoding vWF-cleaving protease also known as ADAMTS-13 that is an active body having the activity of the protease described above was cloned by the present inventors (JP Patent Publication (Kokai) No. 2003-284570). The findings about the molecular structure of ADAMTS-13 are summarized below. The location of a residue number that is numbered from methionine encoded by an initiation codon (ATG) is shown as a rough guide within parentheses (see SEQ ID No. 1).
The domain structure of ADAMTS-13 has a signal peptide preceding a propeptide that ends in a RQRR (SEQ ID NO: 19) sequence as a furin cleavage motif, followed by a metalloprotease domain containing a reprolysin-type zinc-chelating region consisting of HEXXHXXGXXHD (SEQ ID NO: 20) as a consensus sequence (to amino acid residue No. 284 (p285X)); via a disintegrin-like domain as found in snake venom metalloprotease (to amino acid residue No. 386 (W387X)), there exists a first Tspl motif (Tspl-1) consisting of approximately 50 to 60 residues generally considered to be important for molecular recognition (to amino acid residue No. 448 (Q449X)), which continues to a Cys-rich region containing a RGDS (SEQ ID NO: 21) sequence as one of cell adhesion motifs (to amino acid residue No. 580 (T581X)); and, through a spacer domain consisting of approximately 130 amino acid residues with no cysteine residue (to amino acid residue No. 687 (W688X)), additional Tspl motif repeats (Tspl-2 to Tspl-8) follow, which is followed by a CUB domain-1, and -2 that are said to be first found in a complement component C1r or C1s.
By the way, no finding about a major neutralizing epitope region in ADAMTS-13 has been obtained so far. In addition, a convenient diagnostic method for a patient positive for an autoantibody against the protease has not been established.
In light of such circumstances, a first object of the present invention is directed to an invention relating to the identification of a neutralizing epitope present on ADAMTS-13 and a neutralizing/absorbing agent for an antibody thereby proposed, which is mainly intended for an autoantibody.
A second object of the present invention is to provide a method of producing the neutralizing/absorbing agent.
A third object of the present invention is to provide an application of the neutralizing/absorbing agent.
A fourth object of the present invention is to provide a method of producing a full-length or partially modified molecule of vWF-specific cleaving protease, which is obtained by modifying the epitope.
A fifth object of the present invention is to provide an application of the full-length or partially modified molecule of vWF-specific cleaving protease, which is obtained by modifying the epitope.
Up to now, plasmapheresis has been provided as treatment to a patient congenitally deficient in vWF-specific cleaving protease and a patient with acquired production of an antibody against the protease. Therefore, there is a demand for the establishment of replacement therapy with pure vWF-specific cleaving protease such as the protease purified or genetically altered. It has been reported that a familial TTP patient is congenitally deficient in vWF-specific cleaving protease and non-familial TTP is caused by the acquired production of an autoantibody against the protease. Thus, replacement therapy with the protease is preferred for a familial TTP patient (in reality, plasma administration is given to the patient), while a non-familial TTP patient requires the removal of an autoantibody by plasma exchange as well as the supplementation of the protease.
However, in the administration of ADAMTS-13 for supplementation to a patient positive for an autoantibody, an antibody against the protease, that is, an autoantibody, present in blood of the patient neutralizes the administered protease. As a result, the protease loses enzyme activity and has substantial reduction in concentration. However, the use of a neutralizing region identified by a method of determining an epitope for an antibody against ADAMTS-13 disclosed in a previous application (JP Patent Application No. 2002-279924) or identified by the present invention as well as the preparation of a partially modified molecule of a neutralizing epitope region that can be newly identified by Western blotting of competitive inhibition assay used in the present invention allows the administration of the protease to a patient positive for an antibody against the protease; or alternatively, allows the absorption of the antibody by a polypeptide or the like containing the neutralizing region provided by the present invention.